Effect of the transient pharmacological inhibition of Mapk3/1 pathway on ovulation in mice.

Mitogen-activated protein kinase 3/1 (Mapk3/1) pathway is critical for LH signal transduction during ovulation. However, the mechanisms remain incompletely understood. We hypothesized that Mapk pathway regulates ovulation through transcriptional regulation of ovulatory genes. To test this hypothesis we used immature mice superovulated with equine and human chorionic gonadotropins (eCG and hCG) and PD0325901, to inhibit hCG-induced Mapk3/1 activity. Mice received either the inhibitor PD0325901 (25 μg/g, i.p.) or vehicle at 2h before hCG stimulation. Administration of the inhibitor abolished Mapk3/1 phosphorylation in granulosa cells. While vehicle-treated mice ovulated normally, there were no ovulations in inhibitor-treated mice. First, we analyzed gene expression in granulosa cells at 0h, 1h and 4h post-hCG. There was expected hCG-driven increase in mRNA abundance of many ovulation-related genes including Ptgs2 in vehicle-treated granulosa cells, but not (P<0.05) in inhibitor-treated group. There was also reduced mRNA and protein abundance of the transcription factor, early growth response 1 (Egr1) in inhibitor-treated granulosa cells. We then used GRMO2 cell-line to test if Egr1 is recruited to promoter of Ptgs2 followed by chromatin immunoprecipitation with either Egr1 or control antibody. Enrichment of the promoter regions in immunoprecipitants of Egr1 antibody indicated that Egr1 binds to the Ptgs2 promoter. We then knocked down Egr1 expression in mouse primary granulosa cells using siRNA technology. Treatment with Egr1-siRNA inhibited Egr1 transcript accumulation, which was associated with reduced expression of Ptgs2 when compared to control-siRNA treated granulosa cells. These data demonstrate that transient inhibition of LH-stimulated MAPK3/1 activity abrogates ovulation in mice. We conclude that Mapk3/1 regulates ovulation, at least in part, through Egr1 and its target gene, Ptgs2 in granulosa cells of ovulating follicles in mice

Map2k inhibitor treatment does not shutdown the global transcription in granulosa cells.

<p>Treatment of immature mice with single dose of 25μg/g of PD0325901 did not alter relative mRNA abundance of Mapk1 and Mapk3 (A). Expression of known hCG regulated genes like <i>Fshr</i> and <i>Nr5a2</i> (B) as well as hCG induced genes like <i>Scarb1</i> and <i>Pappa</i> (C) were also not altered in PD0325901 treated mice (n = 3mice/group/time point).</p

Inhibition of Mapk3/1 activity and knockdown of Egr1 results in reduced induction of Ptgs2 in primary granulosa cells in vitro.

<p>A) Treatment with PD0325901 decreased Fo+PMA induced increases in the abundance of Ptgs2 protein relative to vehicle treated primary granulosa cells. B) Pre-treatment with Egr1-siRNA inhibited Fo+PMA induced expression of Egr1 when compared to control siRNA treatment in primary granulosa cells in vitro. This was associated with reduced abundance of Pgts2 transcript in Egr1-siRNA treated cells. Fo—forskolin; PMA—phorbol-12-myristate.</p

Inhibition of hCG induced cumulus expansion, oocyte maturation and follicular rupture due to Map2k inhibitor treatment.

<p>Treatment of immature mice with single dose of 25μg/g of PD0325901 resulted in anovulation compared to vehicle treated mice (A) during superovulation (n = 5 mice/ group). This anovulation as evidenced by trapped GV stage oocytes with compact cumulus cells (C&D) inside the preovulatory follicles at 18h hCG (n = 5 mice/ group). Vehicle treated mice showed well-developed corpus luteum (B) evidencing follicular rupture, ovulation and luteinization. CL—corpus luteum; GV—germinal vesicle.</p

Transcriptional regulation of ovulatory genes by Mapk3/1 through Egr1 transcription factor.

<p>Preovulatory LH surge induced Mapk3/1 activity regulates transcription of ovulatory genes such as <i>Ptgs2</i> trough transcription factor Egr1. Inhibition of this LH-induced Mapk3/1 activity by Map2k inhibitor PD0325901 results in anovulation.</p

Map2k inhibitor treatment reduces the expression of hCG induced ovulatory genes.

<p>Treatment of immature mice with single dose of 25μg/g of PD0325901 inhibited the expression of hCG-induced genes involved in follicular rupture like <i>Ptgs2</i>, <i>Pgr</i>, <i>Adamts1</i>; genes involved in cumulus expansion like <i>Has2</i>, <i>Ptx3</i>, <i>Tnfaip6</i>; gene involved in oocyte meiotic maturation like Areg and luteinization like <i>Cebpb</i> (n = 3mice/group/time point). Mapk3/1 regulated LH induced genes like <i>Ptgs2</i>, <i>Ptx3 and Tnfaip6</i> were not regulated by Cebpb.</p

Effect of the Transient Pharmacological Inhibition of Mapk3/1 Pathway on Ovulation in Mice - Fig 6

<p>A) Comparison of expression profile of ovulatory genes in granulosa cells from hCG treated mice and cAMP treated GRMO2 cells. Expression profiles of ovulatory genes like <i>Star</i>, <i>Ptgs2</i> and <i>Egr1</i> were similar at 0h, 1h and 4h after hCG or cAMP treatment in granulosa cells or cultured GRMO2 cells respectively. B) Binding of Egr1 transcription factor to Ptgs2 promoter region (-159 to -33 relative to the transcription start site). Chromatin immunoprecipitation and qPCR using 4h cAMP treated GRMO2 cells showed the enrichment of promoter region containing Egr1 binding site (identified by bioinformatic analysis) in immunoprecipitants of Egr1 antibody indicating that Egr1 binds to Ptgs2 promoter. Rabbit IgG was used as a control antibody.</p

Transient inhibition of hCG induced Mapk 3/1 activity by Map2k inhibitor treatment during ovarian superstimulation protocol.

<p>Granulosa cells collected by follicular puncture from mice treated with PD0325901 showed absence of phosphorylation of Mapk3/1 at Thr302/Tyr204 compared to vehicle treated mice (n = 3 mice/group/time point).</p